I domain chimeric antigen receptor specific to icam-1

ABSTRACT

The present invention relates to chimeric antigen receptors (CARs) specific to ICAM-1 comprising I domain of the α L  subunit of human lymphocyte function-associated antigen 1 (LFA-1). The invention particularly relates to CARs comprising human I domains having different affinities (1 mM to 1 nM Kd) to ICAM-1. CAR T cells comprising human I domain having a low affinity (1 to 200 μM Kd) to ICAM-1 can avoid targeting healthy tissues with basal ICAM-1 expression while simultaneously exhibiting increased potency and long-term efficacy against tumor tissues with high ICAM-1 expression. The present invention also relates to an adoptive cell therapy method for treating cancer by administering the CAR-T cells comprising human I domain to a subject suffering from cancer, whereby the CAR T cells bind to the cancer cells overexpressing ICAM-1 and kill the cancer cells.

This application claims the benefit of U.S. Provisional Application Nos.62/383,139, filed Sep. 2, 2016; and 62/419,817, filed Nov. 9, 2016;which are incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith with thespecification as an ASCII formatted text file via EFS-Web with a filename of Sequence Listing.txt with a creation date of Jul. 17, 2017, anda size of 9.89 kilobytes. The Sequence Listing filed via EFS-Web is partof the specification and is hereby incorporated in its entirety byreference herein.

FIELD OF THE INVENTION

The present invention relates to chimeric antigen receptors specific toICAM-1 comprising human I domain. The invention particularly relates tochimeric antigen receptors comprising human I domains having differentaffinities (1 mM to 1 nM) to ICAM-1.

BACKGROUND OF THE INVENTION

Immunotherapy is emerging as a highly promising approach for thetreatment of cancer. Genetically modifying T cells with CARs is a commonapproach to design tumor-specific T cells. CAR (chimeric antigenreceptor)-T cells targeting tumor-associated antigens can be infusedinto patients (adoptive cell transfer or ACT) representing an efficientimmunotherapy approach. The advantage of CAR-T technology compared withchemotherapy or antibody is that reprogrammed engineered T cells canproliferate and persist in the patient and work like a living drug.

CAR molecules are composed of synthetic binding moieties, typically anantibody-derived single chain fragment variable (svFv) or any nativeantigen-sensing element, fused to intracellular signaling domainscomposed of the TCR zeta chain and costimulatory molecules such as CD28and/or 4-1BB^(1,2). The advantages of CAR mediated targeting include: 1)the provision of activation, proliferation, and survival signals in-cisvia a single binding event, compared to the natural, non-integrated TCRand costimulatory signaling; 2) the ability to bypass the downregulationof MHC by tumor cells through MHC-independent antigen recognition; and3) a reduced activation threshold as well as recognition of tumor cellswith low antigen density enabled by the high affinity interactionbetween CAR and antigen^(3, 4).

The ideal CAR target antigen would be a native, surface-exposed tumorneoantigen that is highly expressed and is undetectable in healthytissues. However, due to the implicit rarity of such antigens, manycommonly targeted solid tumor antigens, are also expressed by non-tumortissues, albeit at lower levels. CAR molecules with high affinity tosuch antigens can lead to collateral targeting of healthy tissuesresulting in on-target, off-tumor toxicity, a major limiting factor tothe progress of CAR T cell therapy to date.

Conventional CARs are constructed using a single-chain antibody format,and are selectively engineered to possess sub- to low nanomolaraffinities for target antigens. However, increased CAR T cellsensitivity may be an advantage only when targeting true tumor antigensor those with the highest levels of restriction^(17, 36). Otherwise,increased sensitivity comes at the price of reduced selectivity withlysis of target-expressing cells in a manner largely insensitive toantigen density¹⁸.

There exists a needs for CARs with improved therapeutic index, i.e.,CARs that can kill tumor while minimizing systemic toxicity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G Show Construction of ICAM-1 Specific CARs with Step-Wise,10⁶-Fold Variations in Affinity and their In Vitro Results.

FIG. 1A: Schematic of LFA-1 in complex with ICAM-1. α and β chains, andmodular domains of LFA-1 integrin are labeled. Metal ions necessary forLFA-1 and ICAM-1 interaction are shown in circles.

FIG. 1B: Structural model of LFA-1 I domain and the N-terminal domain ofICAM-1 (D1) are drawn in ribbon diagram. N and C-termini, and mutationalhot spots are indicated.

FIG. 1C: SPR sensogram of I domain variants binding to immobilized humanICAM-1, except F265S/F292G*, which was flowed over murine ICAM-1(adapted from FIG. 2 of Jin et. al.⁵⁴, and FIG. 1 of Wong et. al⁵⁵).

FIG. 1D: A schematic of the lentivirus vector encoding I domain CAR.LTR=long terminal repeat; SD=splice donor; SA=splice acceptor;ψ⁺=packaging signal; SS=signal sequence; TM=transmembrane; Cyt=cytosolicdomain.

FIG. 1E: Anti-Myc antibody binding to Jurkat T cells transduced withMyc-tagged CARs (TM, F292G, F292A, and WT I domain). NT=non-transduced.

FIG. 1F: Recombinant ICAM-1-Fc binding to CARs expressed in HEK 293Tcells.

FIG. 1G: V-bottom adhesion assay measuring relative binding affinitiesbetween I domain CARs expressed in Jurkat T cells and soluble human(top) and murine (bottom) ICAM-1 (CD54) coated surfaces. n=3; p<0.01for * vs. NT by Dunnett's multiple comparisons test.

FIGS. 2A-2D Show Affinity and Antigen-Density Dependent Activation ofPrimary CAR T Cells In Vitro.

FIG. 2A: Effector to target (E:T) assay for measuring target killing byprimary T cells transduced with different I domain CARs. Each target wasseparately incubated with TM, F292G, F292A or WT CAR T cells at 5:1 E:Tratio. Percent viability was normalized to luminescence from targetcells incubated with NT T cells (n=3, ±=standard deviation (SD)). Avariable slope sigmoidal curve equation was used to fit data. p<0.01for * vs. NT by Dunnett's multiple comparisons test.

FIG. 2B: The best fit values of 50% killing and Hill slope of thesigmoidal equation were plotted against the affinities of I domain CARs.The best fit values with r-square values higher than 0.85 were plotted.

FIG. 2C: ICAM-1 expression in primary T cells in comparison to HeLacells. Grey and black histograms correspond to unlabeled cells and R6.5antibody-labeled cells, respectively.

FIG. 2D: IFN-γ release was measured by ELISA for each CAR T variantafter co-incubation with different target cells for 24 h (n=3). p<0.01for * vs. 8505C/-ICAM-1 by Dunnett's multiple comparisons test.

FIGS. 3A-3C Show Micromolar Affinity CAR T Cells Provide Superior TumorEradication, Suppression of Tumor Relapse, and Survival Benefit.

FIG. 3A: Whole-body luminescence imaging was used to estimate tumorburden in mice infused with different CAR T cell variants 8 dayspost-tumor implantation. No T=mice received no T cells.

FIG. 3B: Mice were treated with CAR T cells 10 days post tumorimplantation. NT=non-transduced T cells.

FIG. 3C: Survival curves of mice receiving different treatments.Log-rank (Mantel-Cox) test P values versus NT are not-significant for NoT and TM, and p=0.008 for F292G, p=0.025 for R6.5, and p=0.0016 forF292A.

FIGS. 4A-4D Show Longitudinal, Concurrent Measurements of Tumor Burden,T Cell Distribution, and Cytokine Release.

FIG. 4A: Schematic of SSTR2-I domain vector.

FIG. 4B: Longitudinal measurements of NOTAOCT uptake by PET/CT (top halfof each panel), and tumor burden by whole body luminescence imaging(bottom half of each panel). Images are representative of four mice ineach cohort. Whole body PET/CT images, taken on the day of maximumtracer uptake, are shown on the far right. Imaging time points areindicated below the bottom panel. For example, 15 represents 15 dayspost tumor xenograft (and 7 days post T cell infusion).

FIG. 4C: Quantification of luminescence and tracer uptake in the lungsof mice treated as indicated. Top Panel: NT (non-transduced) T cells.Bottom level: CARs-F292A.

FIG. 4D: Cytokine levels measured from blood drawn at various timepoints from the same mice in ‘b’ and ‘c’ are plotted (mean±SD, duplicatemeasurements). Top Panel: NT (non-transduced) T cells. Bottom level:CARs-F292A.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, “about” refers to ±10% of the recited value.

As used herein, “adoptive T cell therapy” involves the isolation and exvivo expansion of tumor specific T cells to achieve greater number of Tcells than what could be obtained by vaccination alone. The tumorspecific T cells are then infused into patients with cancer in anattempt to give their immune system the ability to overwhelm remainingtumor via T cells which can attack and kill cancer.

As used herein, “affinity” is the strength of binding of a singlemolecule (e.g., I domain) to its ligand (e.g., ICAM-1). Affinity istypically measured and reported by the equilibrium dissociation constant(K_(D) or Kd), which is used to evaluate and rank order strengths ofbimolecular interactions.

As used herein, a “chimeric antigen receptor (CAR)” means a fusedprotein comprising an extracellular domain capable of binding to anantigen, a transmembrane domain derived from a polypeptide differentfrom a polypeptide from which the extracellular domain is derived, andat least one intracellular domain. The “extracellular domain capable ofbinding to an antigen” means any oligopeptide or polypeptide that canbind to a certain antigen. The “intracellular domain” means anyoligopeptide or polypeptide known to function as a domain that transmitsa signal to cause activation or inhibition of a biological process in acell.

As used herein, a “domain” means one region in a polypeptide which isfolded into a particular structure independently of other regions.

As used herein, “integrin” or “integrin receptor” (used interchangeably)refers to any of the many cell surface receptor proteins, also referredto as adhesion receptors which bind to extracellular matrix ligands orother cell adhesion protein ligands thereby mediating cell-cell andcell-matrix adhesion processes. Binding affinity of the integrins totheir ligands is regulated by conformational changes in the integrin.Integrins are involved in physiological processes such as, for example,embryogenesis, hemostasis, wound healing, immune response andformation/maintenance of tissue architecture. Integrin subfamiliescontain a beta-subunit combined with different alpha-subunits to formadhesion protein receptors with different specificities.

“Intercellular adhesion molecule-1” or “ICAM-1”, i.e. GenBank AccessionNos. NM_000201, NP_000192, is the ligand for α_(L)β₂ integrin, and itsN-terminal domain (D1) binds to the α_(L) I domain through thecoordination of ICAM-1 residue Glu-34 to the MIDAS metal. ICAM1 istypically expressed on endothelial cells and cells of the immune system.ICAM1 binds to integrins of type α_(L)β₂ and α_(M)β₂. ICAM-1 isupregulated in several carcinomas and the associated stroma²⁴ as well asin inflammatory conditions²⁵. Aside from diseased tissues, ICAM-1 isbasally expressed in several cell types including endothelial cells,immune cells, and some epithelial cells²⁵.

“Lymphocyte function-associated antigen-1”, “LFA-1”, “α_(L)β₂ integrin”or “CD18/CD11a” refers to a member of the leukocyte integrin subfamily.LFA-1 is found on all T-cells and also on B-cells, macrophages,neutrophils and NK cells and is involved in recruitment to the site ofinfection. It binds to ICAM-1 on antigen-presenting cells and functionsas an adhesion molecule.

As used herein, “I domain” refers to the inserted or I domain of theα_(L) subunit of LFA-1, and is an allosteric mediator of ligand bindingto LFA-1. I domain is a native ligand of ICAM-1. The ligand binding siteof the I domain, known as a metal ion-dependent adhesion site (MIDAS),exists as two distinct conformations allosterically regulated by theC-terminal α7 helix. A wild-type (WT) I domain encompasses amino acidresidues 130-310 of the 1145 amino acid long mature α_(L) integrinsubunit protein (SEQ ID NO: 1, which is the amino acid residues 26-1170of GenBank Accession No. NP_002200). All numbering of amino acidresidues as used herein refers to the amino acid sequence of the matureα_(L) integrin (SEQ ID NO: 1), wherein residue 1 of SEQ ID NO: 1corresponds to residue 26 of the sequence of GenBank Accession No.NP_002200.

As used herein, a “tumor antigen” means a biological molecule havingantigenicity, expression of which causes cancer.

DESCRIPTION

The present invention provides chimeric antigen receptors targetingICAM-1, which is a broad tumor biomarker, using its physiologicalligand, LFA-1. The inventor has constructed a panel of affinity-variantCARs that comprise human I domain; the CARs having 1 mM to 1 nM affinityto ICAM-1. The present invention provides ICAM-1-specific CARS withbroad anti-tumor applicability. CAR T cells comprising I domain havingmicromolar affinity targeting ICAM-1 have improved efficacy and safetyover conventional CARS, as they are capable of lysing cellsoverexpressing target antigens while sparing normal cells with muchlower densities.

The present invention is directed to a chimeric antigen receptor fusionprotein comprising from N-terminus to C-terminus: (i) a human I domainof the α_(L) subunit of lymphocyte function-associated antigen-1, (ii) atransmembrane domain, (iii) at least one co-stimulatory domains, and(iv) an activating domain.

The CAR of the present invention comprises (i) a human I domain thatbinds specifically to ICAM-1. I domain specific to ICAM-1 can be builtusing the I domain derived from LFA-1 (FIGS. 1A and 1B). Variousactivating point mutations in the I domain are localized outside of thebinding interface that includes a region known as the metal-iondependent adhesion site (MIDAS) (FIG. 1B). Mutants containing thestep-wise elevation of I domain affinity to ICAM-1 from 1 mM to 1 nM canbe obtained by screening a library of mutants for their higher bindingto ICAM-1 coated surface, beads, or cells. For example, differentaffinity mutants can be isolated using a yeast display system (see Jinet al.²⁷). Affinity is first measured by surface plasmon resonance(e.g., Biacore) to assess 1:1 binding affinity between I domain andICAM-1. Affinity of ICAM-1 to CAR expressed on cells can be measured byflow cytometry and using the Langmuir isotherm equation. Likewise,Scatchard analysis can be performed to estimate CAR affinity bymeasuring the amounts of free and cell-surface bound ligand (in thiscase, radio- or fluorescence-labeled ICAM-1).

Table 1 shows measured affinities of LFA-1 I domains of wild type andmutants to ICAM-1. A majority of mutations are changing hydrophobicbulky side chains (F, L, I) into more hydrophilic (A, S, T), therebydisrupting the structure of more compact, low affinity I domainconformation. For example, substitution of Phe-292 located in theC-terminal α7-helix with Ala (F292A) and Gly (F292G) provides toaffinities (K_(D)) of ˜20 μM and 0.1 respectively (Table 1). Thecombination of F292G with another comparably activating mutation inPhe-265 (F265S/F292G) provides an affinity of 6 nM, approximately200,000-fold higher than the wild-type (WT) I domain (K_(D)=1.5 mM)(FIG. 1C). To lock the C-terminal α7-helix of F265S/F292G in the openposition (FIG. 1A), Gly-311 can be replaced with Cys (G311C) in theF265S/F292G mutant (F265S/F292G/G311C, dubbed triple mutant or TM) toform a disulfide bond with the naturally unpaired Cys-125 (Table 1).Therefore, the monovalent affinities of individual I domain variants forICAM-1 can be designed to span approximately six orders of magnitude(K_(D)˜1 nM to 1 mM), as measured by surface plasmon resonance (SPR) orestimated by flow cytometry (FIG. 1C, Table 1). The mutants in Table 1are for illustration purpose only; the CARs of the present invention arenot limited to these specific mutants. Mutants that have other mutationsand have affinities to ICAM-1 between 1 mM to 1 nM can be made, tested,and selected according to methods known to a skilled person.

TABLE 1 Sequence of Name SEQ ID NO: 1 Affinity Wild-type (WT) G128-G3111.5 mM* I288N G128-G311 202 μM** I309T G128-G311 127 μM** L295AG128-G311 37 μM** F292A G128-G311 20 μM* F292S G128-G311 1.24 μM** L289GG128-G311 196 nM** F292G G128-G311 119 nM* F265S G128-G311 145 nM*F265S/F292G (DM) G128-G311 6 nM* F265S/F292G/G311C (TM) E124-S313 ~1 nM*R6.5 scFv 10 nM*** *SPR measurements; **Estimated from flow cytometrymean fluorescence intensity (MFI) values of ICAM-1-Fc binding to yeastcells expressing I domain variants⁵. The equation used was Kd (M) =0.00175*exp(−0.1542*MFI); ***Estimated from titrated R6.5 antibodybinding to HeLa cells³⁴.

In one embodiment, the CAR of the present invention comprises I domainthat is a wild-type human I domain, a mutant of wild-type human I domainhaving 1 to 3 amino acid mutations, or a sequence having at least 95%,or at least 96% identity, or at least 97% identity, or at least 98%identity, or at least 99% identity to the sequence of the wild-type Idomain or the mutant, having an affinity of binding human ICAM-1 of 1 mMor stronger. In one embodiment, the mutant may have one or moremutations at the amino acid residue 265, 288, 289, 292, 295, 309, or 311of the wild-type I domain. For example, the mutant may have one or moremutations of I288N, I309T, L295A, F292A, F292S, L289G, F292G, F265S,F265S/F292G, or F265S/F292G/G311C, of the wild-type I domain. In oneembodiment, combining two I domain mutations produces a mutant with ahigher affinity than that of each parent mutant. For example, combiningtwo mutants each having about 100 μM Kd typically produces a mutanthaving about 1 to about 10 μM Kd range. F292G is a very potent pointmutation; combining F292G with other mutations increases I domainaffinity to ICAM-1 to stronger than 100 nM Kd. The above numbering ofthe amino acid residues is in reference to the mature amino acidsequence of SEQ ID NO: 1, and residue number 1 corresponds to the aminoacid residue 26 of GenBank Accession No. NP_002200.

In one embodiment, the CAR of the present invention comprises I domainthat binds ICAM-1 at an affinity between 1 mM to 1 nM Kd, preferably1-200 μM Kd or 1-20 μM Kd.

In one embodiment, the CAR of the present invention comprises I domainthat binds to ICAM-1 at an affinity between about 120 nM to about 1 nMKd, e.g., F292G, F265S, F265S/F292G, and F265S/F292G/G311C.

In one embodiment, the CAR of the present invention comprises I domainthat binds to ICAM-1 at an affinity between about 20 μM to about 120 nMKd, e.g., F292A, F292S, and I289G.

In one embodiment, the CAR of the present invention comprises I domainthat binds to ICAM-1 at an affinity between about 200 μM to about 20 μMKd, e.g., I288N, I309T, L295A, and F292A.

In one embodiment, the CAR of the present invention comprises I domainthat binds to ICAM-1 at an affinity between about 1 μM to about 100 μMKd, e.g., L296A, F292A and F292S.

In one embodiment, the CAR of the present invention comprises I domainthat binds to ICAM-1 at an affinity between about 1 mM to about 200 μMKd, e.g., wild-type and I288N.

In one embodiment, the CAR of the present invention comprises I domainthat binds to ICAM-1 at an affinity between about 1 mM to about 100 μMKd, e.g., wild-type, I288N, and I309T. The affinities in the aboveembodiments refer to the interaction between I domain and ICAM-1 insolution.

One advantage of using human I domain in CAR construction is that humanI domain binds murine ICAM-1 with comparable affinity to human ICAM-1 (2nM vs. 6 nM respectively). Cross-reactivity with its murine homologueenables examination of on-target, off-tumor toxicity of I domain CAR Tcells concurrently with on-target, on-tumor efficacy in preclinicalmouse models with human tumor xenografts. This is an advantage of humanI domain in predicting clinical toxicity in a preclinical model. Incomparison, the R6.5 scFv (derived from the mouse hybridoma clone,R6.533) has a Kd of 10 nM for human ICAM-1 (Table 1) but does notcross-react with murine ICAM-1.

The CAR of the present invention comprises (ii) a transmembrane domainwhich spans the membrane. The transmembrane domain may be derived from anatural polypeptide, or may be artificially designed. The transmembranedomain derived from a natural polypeptide can be obtained from anymembrane-binding or transmembrane protein. For example, a transmembranedomain of a T cell receptor α or β chain, a CD3 zeta chain, CD28,CD3-epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64,CD80, CD86, CD134, CD137, ICOS, CD154, or a GITR can be used. Theartificially designed transmembrane domain is a polypeptide mainlycomprising hydrophobic residues such as leucine and valine. In preferredembodiments, the transmembrane domain is derived from CD28 or CD8, whichgive good receptor stability.

The CAR of the present invention comprises (iii) one or moreco-stimulatory domains selected from the group consisting of human CD28,4-1BB (CD137), ICOS-1, CD27, OX 40 (CD137), DAP10, and GITR (AITR). Inembodiment, the CAR comprises two co-stimulating domains of CD28 and4-1BB.

The endodomain (the activating domain) is the signal-transmissionportion of the CAR. After antigen recognition, receptors cluster and asignal is transmitted to the cell. The most commonly used endodomaincomponent is that of CD3-zeta (CD3 Z or CD3ζ), which contains 3 ITAMs.This transmits an activation signal to the T cell after antigen isbound. CD3-zeta may not provide a fully competent activation signal andadditional co-stimulatory signaling may be needed. For example, one ormore co-stimulating domains can be used with CD3-Zeta to transmit aproliferative/survival signal.

The CAR of the present invention may comprise a signal peptideN-terminal to the I domain so that when the CAR is expressed inside acell, such as a T-cell, the nascent protein is directed to theendoplasmic reticulum and subsequently to the cell surface, where it isexpressed. The core of the signal peptide may contain a long stretch ofhydrophobic amino acids that has a tendency to form a singlealpha-helix. The signal peptide may begin with a short positivelycharged stretch of amino acids, which helps to enforce proper topologyof the polypeptide during translocation. At the end of the signalpeptide there is typically a stretch of amino acids that is recognizedand cleaved by signal peptidase. Signal peptidase may cleave eitherduring or after completion of translocation to generate a free signalpeptide and a mature protein. The free signal peptides are then digestedby specific proteases. As an example, the signal peptide may derive fromhuman CD8 or GM-CSF, or a variant thereof having 1 or 2 amino acidmutations provided that the signal peptide still functions to cause cellsurface expression of the CAR.

The CAR of the present invention may comprise a spacer sequence as ahinge to connect I domain with the transmembrane domain and spatiallyseparate antigen binding domain from the endodomain. A flexible spacerallows to the binding domain to orient in different directions to enableits binding to a tumor antigen. The spacer sequence may, for example,comprise an IgG1 Fc region, an IgG1 hinge or a CD8 stalk, or acombination thereof. A human CD28 or CD8 stalk is preferred.

The present invention provides a nucleic acid encoding the CAR describedabove. The nucleic acid encoding the CAR can be prepared from an aminoacid sequence of the specified CAR by a conventional method. A basesequence encoding an amino acid sequence can be obtained from theaforementioned NCBI RefSeq IDs or accession numbers of GenBenk for anamino acid sequence of each domain, and the nucleic acid of the presentinvention can be prepared using a standard molecular biological and/orchemical procedure. For example, based on the base sequence, a nucleicacid can be synthesized, and the nucleic acid of the present inventioncan be prepared by combining DNA fragments which are obtained from acDNA library using a polymerase chain reaction (PCR).

The nucleic acid encoding the CAR of the present invention can beinserted into a vector, and the vector can be introduced into a cell.For example, a virus vector such as a retrovirus vector (including anoncoretrovirus vector, a lentivirus vector, and a pseudo type vector),an adenovirus vector, an adeno-associated virus (AAV) vector, a simianvirus vector, a vaccinia virus vector or a Sendai virus vector, anEpstein-Barr virus (EBV) vector, and a HSV vector can be used. As thevirus vector, a virus vector lacking the replicating ability so as notto self-replicate in an infected cell is preferably used.

For example, when a retrovirus vector is used, the process of thepresent invention can be carried out by selecting a suitable packagingcell based on a LTR sequence and a packaging signal sequence possessedby the vector and preparing a retrovirus particle using the packagingcell. Examples of the packaging cell include PG13 (ATCC CRL-10686),PA317 (ATCC CRL-9078), GP+E-86 and GP+envAm-12, and Psi-Crip. Aretrovirus particle can also be prepared using a 293 cell or a 293T cellhaving high transfection efficiency. Many kinds of retrovirus vectorsproduced based on retroviruses and packaging cells that can be used forpackaging of the retrovirus vectors are widely commercially availablefrom many companies.

The present invention provides T cells or natural killer cells (NKcells) modified to express the CAR as described above. CAR-T cells orCAR-NK cells of the present invention bind to ICAM-1 via the I domain ofCAR, thereby a signal is transmitted into the cell, and as a result, thecell is activated. The activation of the cell expressing the CAR isvaried depending on the kind of a host cell and an intracellular domainof the CAR, and can be confirmed based on, for example, release of acytokine, improvement of a cell proliferation rate, change in a cellsurface molecule, killing target cells, or the like as an index.

T cells or NK cells modified to express the I domain-CAR can be used asa therapeutic agent for a disease. The therapeutic agent comprises the Tcells expressing the I domain-CAR as an active ingredient, and mayfurther comprise a suitable excipient. Examples of the excipient includepharmaceutically acceptable excipients known to a person skilled in theart.

The present invention further provides an adoptive cell therapy methodfor treating cancer. The method comprises the steps of: administeringthe CAR-T cells or CAR-NK cells of the present invention to a subjectsuffering from cancer, wherein the cancer cells of the subjectoverexpress ICAM-1, and the CAR-T cells or CAR-NK cells bind to cancercells to kill the cancer cells. “Overexpress”, as used herein, refers tocancer cells have surface expression of ICAM-1 at least 10⁵ moleculesper cell. In one embodiment, the CAR comprises I domain having anaffinity to ICAM-1 between about 1 to about 1000 preferably betweenabout 1 to about 200 or about 1 to about 20 μM. Cancers suitable to betreated by the present invention include, but not limited to thyroidcancer, gastric cancer, pancreatic cancer, and breast cancer.

By functionally investigating CAR affinities spanning step-wise across a10⁶-fold range, concurrently with target cells with varying levels ofantigen expression, the inventor systematically examined the influenceof CAR affinity and antigen density on T cell efficacy in vitro and invivo. T cell activation status in vitro, as measured by CD25, cytokinerelease, and cytotoxicity, was dependent on affinity and target antigendensity, resulting in more potent T cell activation and target killingwith increases in CAR affinity and antigen density. The activationthreshold of nanomolar affinity CAR T cells (TM, F292G) was lessdependent on antigen density compared to the micromolar affinity CAR Tcells (F292A), reacting to antigen density as low as 10⁴ molecules/cell.In contrast, F292A CAR T cells rapidly lost the ability to lyse cellsexpressing target antigens below 10⁵ molecules/cell. Millimolar affinityCAR T cells (wide-type, WT) were largely unreactive to target cells withlow to moderate levels of antigen, requiring a threshold antigen densityof 10⁶ molecules/cell for detectable activation, cytokine release, andtarget lysis to occur.

Table 2 shows a range of desired affinities of I domain-comprising CARTcells to ICAM-1, for targeting cells with specified ICAM-1 antigendensity.

TABLE 2 I CAM-1 Density (molecules/cells) Suitable I Domain Affinity <10⁴ about 120 nM-1 nM (e.g., TM, F292G) 10⁴-10⁵ about 20 μM-120 nM(e.g., F292S, F265S) 10⁵-10⁶ about 200 μM-20 μM (e.g., F292A) ≥10⁶ about1.5 mM-200 μM (e.g., WT)

The quantitative harmony between CAR affinity and anti-tumor potency invitro is discordant with quantitative in vivo observations wherebymicromolar affinity (1-200 μM or 1-20 μM) CAR-T cells or CAR-NK cellsare superior to higher affinity CAR-T cells or CAR-NK cells as measuredby the rate of expansion at the tumor site, the rate of tumoreradication, frequency of tumor relapse, and levels of on-target,off-tumor toxicity.

The ability of I domain CAR-T cells or CAR-NK cells to cross-react withmurine ICAM-1 allows for a rigorous and simultaneous assessment of theefficacy of CAR-T cells or CAR-NK against human tumor cells andon-target, off-tumor toxicity against murine ICAM-1 on healthy tissues.By simultaneous expression of a reporting gene, human somatostatinreceptor 2 (SSTR2), and I domain CAR on T cells followed by longitudinalposition emission tomography (PET) imaging, in vivo spatiotemporalmapping of adoptively transferred T cells can be achieved.

Onset of toxicity appears to be dependent on CAR affinity andtumor-burden, as demonstrated by the uniform fatalities in mice treatedwith the highest affinity (TM) CAR T cells, the increased rate oftoxicity observed in F292G CAR-treated mice with larger tumor burden,and the absence of detectable toxicity after treatment with micromolaraffinity F292A CAR T cells.

CARs comprising high affinity mutants (about 120 nM-1 nM) have highpotency and they are capable to bind T cells with low ICAM density ofless than 10⁴ per cell.

CARs possessing affinities in the micromolar range (e.g. about 1-200 μMKd) minimize off-tumor toxicity against basally expressed antigens innormal tissues, and also increases therapeutic index, in comparison withCARs having affinities in the nanomolar range (e.g., about 1-200 nM Kd).CAR T cells with target affinities in the micromolar range can avoidtargeting healthy tissue with basal antigen expression whilesimultaneously exhibiting increased potency and long-term efficacyagainst tumor tissue with high target expression. The micromolaraffinity CAR (such as F292A-I domain) enables T cells to neglect tissuesexpressing less than 10⁵ molecules/cell, a threshold which anaplasticthyroid tumors surpass yet healthy tissues typically do not. Engagementof target antigen by nanomolar affinity CAR T cells (e.g., TM, F292G,and R6.5 CAR) may result in an unnaturally slow off rate, deviating fromtransient and dynamic nature of interactions natively found between TCRsand pMHCs⁴⁸. High affinity and avidity interactions by CAR can reduce Tcells' propensity for serial killing, potentially causing exhaustion orincreased susceptibility to activation-induced cell death⁴⁹. AlthoughCAR T cells with nanomolar affinity to ICAM-1 may work, they may beoperating sub-optimally and may be more prone to exhaustion andexcessive cytokine release, ultimately facilitating off-tumor toxicityor tumor relapse.

The following examples further illustrate the present invention. Theseexamples are intended merely to be illustrative of the present inventionand are not to be construed as being limiting.

EXAMPLES Materials and Methods Example 1. Cell Lines and Primary HumanLymphocytes

Human dermal microvascular endothelial cells (HMEC-1) were obtained fromthe Center for Disease Control and were cultured in MCDB 131 medium(Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS,Atlanta Biologicals), 10 mM L-alanyl-L-glutamine dipeptide (Gibco), 100units/ml Penicillin-Streptomycin (Pen-strep), 1 μg/ml hydrocortisome (MPBiomedicals), and 10 ng/ml recombinant human epidermal growth factors(Invitrogen). Mouse brain microvascular endothelial cells (bEnd.3, ATCC)were maintained in Advanced Dulbecco's Modified Eagle Medium (ADMEM,Invitrogen) supplemented with 4 mM L-glutamine, 100 units/ml Pen-strep,and 10% FBS. HeLa cells (ATCC) were cultured in ADMEM containing 10%FBS, 2 mM L-glutamine, and 100 units/ml Pen-strep. 8505C cells (DSMZ)were cultured in RPMI-1640 medium (Invitrogen) containing 10% FBS, 2 mML-glutamine, and 100 units/ml Pen-strep. HMEC-1, bEnd.3, HeLa, and 8505ccells were transduced with lentivirus encoding FireflyLuciferase-F2A-GFP (Biosettia) and sorted based on fluorescence.

Human peripheral blood was obtained from healthy volunteer donors byvenipuncture. Peripheral blood mononuclear cells were isolated usingFicoll-Paque PLUS (GE Healthcare) and cultured in Optimizer CTS T-cellExpansion SFM (Thermo) supplemented with 5% human AB serum (Sigma), 2 mML-alanyl-L-glutamine dipeptide, and 30 IU/ml human IL-2 (Cell Sciences)(T cell culture medium). Non-adherent cells were removed after 24 h andenriched for T cells with Dynabeads CD3/CD28 T cell expander (Thermo) ata 2:1 bead to T cell ratio. Dynabead-bound T cells were subsequentlycultured in IL-2 containing media at a density of 1×10⁶ cells/ml. Allcells were incubated at 37° C. in a 5% CO₂ humidified incubator.

Example 2. Construction of I Domain CAR Vector

Genetic sequences encoding LFA-1 I domains of varying affinities toICAM-1 were derived from a previous study²⁷. I domain variants werefused at the C-terminus directly to the CD8 hinge, CD28 transmembranedomain, and the intracellular portions of the 3^(rd) generation CARarchitecture incorporating the cytoplasmic domains of CD28, CD137, andCD3ζ. The complete CAR inserts were then subcloned into a pLentibackbone²⁹. A reporter gene for CAR T cell imaging, SSTR2, was linked toI domain at the N-terminus using a ‘ribosome skipping’ porcineteschovirus-1 2A (P2A) sequence to ensure comparable production of CARand SSTR2 from the same mRNA.

Example 3. Lentivirus Production and Transduction of T Cells

Lentivirus was produced by transiently transfecting HEK 293T cells usingcalcium phosphate. Briefly, 10 μg of transfer gene, 7.5 μg of pCMV-dR8.2(Addgene) and 5 μg of pCMV-VSVG (Addgene) were mixed and incubated with2 M CaCl₂ followed by 2×HBSS. Resulting solutions were added dropwise to10 cm² cell culture dishes seeded with 3.2×10⁶ HEK 293T cells in 10 mlDMEM 24 h previously. Transfection media was replaced after 6 h. Mediacontaining lentivirus was harvested at 48 and 72 h post transfection,filtered through 0.45 μm filters, and concentrated byultracentrifugation at 75,000×g for 2 h at 4° C. Lentivirus was thenresuspended in serum containing media and frozen at −80° C. Human Tcells were transduced 24-72 h post activation with anti-CD3/CD28Dynabeads either by spinfection (1,000 g for 1 h at 32° C.) or byovernight incubation with lentivirus. T cells were transduced once more24 h after the first transduction. During and following transductions,media containing IL-2 was replaced with media containing human IL-7 (10ng/ml) and IL-15 (5 ng/ml) (Peprotech). Jurkat T cells were transducedby a single overnight incubation with lentivirus.

Example 4. In Vitro Target Cell Killing Assay

2×10⁵ target cells (HMEC-1, bEnd.3, HeLa, and 8505c) stably transducedto express GFP and firefly luciferase were co-cultured with eithernon-transduced or I domain CAR T cells at varying effector to targetratios (E:T). In certain conditions, the ICAM-1 gene was disrupted in8505C cells using CRISPR/Cas9 (Santa Cruz, #sc-400098; denoted as8505C/-ICAM-1) or, alternatively, 8505C cells were exposed to 1 μg/mllipopolysaccharide (LPS; Escherichia coli 026:B6, Sigma) for 12 h toinduce overexpression of ICAM-1 (denoted as 8505C/LPS). Co-cultures werecarried out in T cell culture medium containing 150 μg/ml D-Luciferin(Gold Biotechnology) and no cytokine supplementation. Luminescence wasmeasured using a plate reader (TECAN infinite M1000 PRO) with readingsin each E:T condition normalized to the non-transduced T cell:targetco-culture controls.

Example 5. 8505C Mouse Model, Whole-Body Tumor Imaging, and SerumCytokine Analysis

7.5×10⁵ 8505C cells were injected into NSG mice via tail vein. 1-3×10⁶ Tcells were injected via tail vein 8-10 days after tumor cell injection.Injection timing was chosen based on prior studies with R6.5 CAR T cellswhich demonstrated tumor elimination using similar CAR dosages at up to10-days post xenograft²⁹. Luminescence imaging of tumor xenografts inlive mice was performed using a whole body optical imager (In-VivoExtreme, Bruker). Mice were anesthetized with 2% isoflurane in 2 L/minO₂. Tumor burden was quantified through integration of luminescence overchest cavity and the entire mouse body. For serum cytokine analysis,50-100 μl of blood was collected via tail-vein into Eppendorf tubes onice. Plasma was immediately isolated after removing cell pellet bycentrifugation at 2,000 g for 10 min at 4° C., and stored at −80° C.Human cytokines (GM-CSF, IL-2, IL-6, IFN-γ, TNF-α, CXCL10) were measuredin duplicate using Bio-Plex MAGPIX (Bio Rad) according to themanufacturer's instructions.

Example 6. Ex Vivo Cellular Analysis

Tumor xenografts were resected from mice at appropriate time points.Resected tumors were diced and flushed through 80 μm cell strainers toyield single cell suspensions. Red blood cells were lysed by incubationwith 1×RBC lysis buffer (eBiosciences). Remaining cells were washed,re-suspended in 1×HBSS containing 2% normal goat serum, and blocked withmouse IgG at 2 μg/ml for 10 min. This was followed by staining with 1μg/ml Propidium Iodide (Invitrogen) in combination with 2 μg/ml mouseanti-human CD3-Alexa Fluor 647 (Biolegend) or 2 μg/ml rabbitanti-c-myc-Alexa Fluor 647 (Biolegend). Resulting cells were acquired ona Gallios flow cytometer (Beckman Coulter). Initial flow cytometry gateswere determined based on live cell gating (Propidium Iodide negative).

Example 7. ICAM-1 and CAR Expression Quantification

ICAM-1 expression on various cell lines was determined using a mouseanti-human R6.5 monoclonal antibody (10 μg/ml) obtained from hybridoma(ATCC). I domain CAR expression on T cells was detected using 2 μg/mlrabbit anti-c-myc-Alexa Fluor 647 (Biolegend). I domain Jurkat T cellvariants were incubated with 10 μg/ml recombinant human ICAM-1 fused tohuman Fcγ (R&D Systems). Cells were then washed and resuspended in 1μg/ml rabbit anti-human PE (Santa Cruz Biotechnology) prior to flowcytometry analysis.

Example 8. In Vitro Measurement of IFN-γ

Target cells were washed and suspended at 1×10⁶ cells/ml in T cellculture medium without cytokines. 100 μl of each target cell was addedin triplicate to a 96-well round bottom plate (Corning). T cellsresuspended at 5×10⁶ cells/ml in T cell culture medium were combinedwith target cells in appropriate wells. Plates were incubated at 37° C.for 24-48 h. After incubation, supernatants were collected for ELISA todetect IFN-γ (Biolegend).

Example 9. CD25 and CD69 Staining

Jurkat cells modified with I domain CARs were co-cultured with targetcells at an effector to target ratio of 1:1 (1×10⁵ effectors: 1×10⁵targets) in a 96-well plate. The plate was incubated at 37° C. for 6 h.After incubation, cells were washed prior to labelling with 2 μg/mlanti-human CD25-allophycocyanin (APC; Biolegend) for 30 min on ice.After incubation, samples were washed and analyzed by flow cytometry. Asan alternative to ICAM-1 expressing cells, we also used microbeadscoated with known amounts of ICAM-1. 1×10⁶ sulfate latex microbeads (8□m, ThermoFisher Scientific) were resuspended in 100 uL of PBScontaining indicated amounts of human or murine recombinant ICAM-1-Fcγ(R&D Systems) conjugated with Cy5.5 (Sulfo-Cyanine5.5 NHS ester,Lumiprobe) overnight at room temperature with gentle mixing.Protein-labeled particles were pelleted and resuspended in fresh PBScontaining 0.1 M glycine pH 7.4 for 1 h, while supernatant was used tomeasure bead adsorption efficiency by fluorescence (TECAN infinite M1000PRO). After saturation of bead surface with glycine, beads were pelletedand resuspended in PBS containing 5 mM MgCl₂. Jurkat cells modified witheach I domain CAR variant were incubated with ICAM-1-bound latex beadsat 1:3 (cell:bead) ratio overnight at 37° C. Cells were then collected,labeled with 2 μg/ml anti-human CD69-APC (Biolegend) for analysis byflow cytometry.

Example 10. V-Bottom Adhesion Assay

V-bottom 96-well plates (Corning) were coated with either murine orhuman ICAM-1-Fcγ (10 μg/ml in PBS, pH 7.4) or 2% BSA at 4° C. overnight.The plates were then blocked with 2% BSA for at 37° C. 1 domain CAR Tclones were first stained with CellTracker Orange according tomanufacturer's protocol and then added to ICAM-1-coated wells in 50 μlof PBS containing 5 mM MgCl₂ and 1% BSA. Plates were immediatelycentrifuged at 200 g for 15 min at room temperature. Nonadherent cellsthat accumulated at the bottom of the V-bottom plates were quantified bya fluorescence plate reader (TECAN infinite M1000 PRO), Cell binding toICAM-1 was calculated from the fluorescence intensity values ofexperimental measurements (F_(CAR) and F_(NT)) and normalized to thefluorescence from the wells coated with BSA alone (F_(BSA)):100×((F_(BSA)−F_(CAR))/F_(BSA))/((F_(BSA)−F_(NT))/F_(BSA)).

Example 11. Labeling of ¹⁸F-NOTA-Octreotide (NOTAOCT)

NOTAOCT (1,4,7-Triazacyclononane-1,4,7-triacetic acid-octreotide³⁰, GMPgrade) was obtained as a 1 mg lyophilized powder (cat #9762, ABXPharmaceuticals). The NOTAOCT vial content was diluted with 18 MW waterto 200 μl (5 mg/ml solution) and stored at 4° C. as a stock solution.For chelation of NOTA with Fluorine-18³¹, 5 μl of NOTAOCT was added to10 μl of 0.1 M sodium acetate, pH 4, 6 μl of 2 mM AlCl3, and 100 μlcontaining ˜30 mCi ¹⁸F. The solution was immediately placed in aThermomixer (Eppendorf) at 100° C. and incubated for 15 minutes followedby cooling to room temperature and dilution in 15 ml ddH₂O. A Sep-Paklight C18 column was regenerated in 3 ml 100% ethanol and washed twicein 5 ml ddH₂O with an observed flow rate of 10 drops per minute. NOTAOCTwas then loaded to the Sep-Pak column, which was later washed in 15 ml18 MW water to eliminate any remaining free ¹⁸F. Trapped NOTAOCT waseluted from the column using 300 μl of ethanol and diluted to 1.5 mlwith PBS for injection, providing the final product in ˜15% ethanolisotonic, injectable solution. The eluent was passed through 0.2 μmfilter. The purity of the final product was checked by reverse phaseHPLC.

Example 12. PET/CT Imaging

Registered CT images were acquired using a micro-PET/CT scanner (Inveon,Siemens) at 1-2 h post NOTAOCT injection. Projection data was acquiredin a cone-beam geometry with approximately 1 s steps at 1 degree angularincrements. At least 10 million coincidence events were acquired for PETper study using a 250 to 750 keV energy window and a 6 ns timing window.A reference tube containing 100 μl of a 10% ID/cm³ equivalent dose forquantification of NOTATOC uptake in vivo. To compute NOTAOCT uptakewithin mouse lungs, ellipsoids were drawn separately on the left andright sides of lungs to enclose the majority of their footprint. The %ID/cm³ values, computed relative to the counts obtained in the referencetube, were approximated to a standard uptake value (SUV³²) by dividing %ID/cm³ by four, assuming injection efficiency of 100% and 25 g of bodyweight. Visualization and analyses of PET/CT images were performed usingAMIDE software (http://amide.sourceforge.net).

Example 12. Histology

After euthanasia, mouse lungs were perfused via trachea with 4%paraformaldehyde, and each of five lobes were separated post fixationand embedded in paraffin. Tissues were cut to produce 5 μm sections(Microtome, Leica). Paraffin embedded sections were stained withhematoxylin and eosin (H&E) or hematoxylin only for CD3 and GFPimmunostaining (performed by HistoWiz, Inc.). Histological analysis wasperformed by an experienced pathologist.

Results Statistical Analysis

One-way ANOVA, Dunnett's multiple comparisons test, and unpairedStudent's t-test were performed using Prism (GraphPad) on dataindicated.

Example 13. ICAM-1 Specific CAR T Cells with 10⁶-Fold, Step-WiseVariation in Affinity

CAR constructs specific to ICAM-1 were built using the I domain derivedfrom LFA-1 (FIGS. 1A-B; Table 1), according to Jin et al²⁷ and U.S. Pat.No. 8,021,668.

To test whether the mutant I domain affinities correlate with CARaffinities, HEK 293T and Jurkat T cells were transduced with lentivirusencoding 3^(rd) generation CARs containing TM, F292G, F292A, or WT Idomain, and assayed for ICAM-1 binding. A myc tag was appended to theN-terminus of each I domain variant to aid measurement of CAR expression(FIGS. 1D-E). To avoid background ICAM-1 binding to endogenous LFA-1 inJurkat T cells, CAR affinity for ICAM-1 was estimated using the I domainCAR-transduced HEK 293T cells. The level of recombinant human ICAM-1binding to I domain CAR-expressing HEK 293T cells correlated withsolution affinity measurements, with TM exhibiting the strongestbinding, followed by F292G and F292A, and no detectable binding to WTcompared to non-transduced (NT) T cells (FIG. 1F). Differential CARaffinities for ICAM-1 and cross-reactivity with murine ICAM-1 were alsoexamined by measuring cell adhesion to V-bottom plates coated withrecombinant human or murine ICAM-1 (FIG. 1G). Jurkat cells transducedwith TM and F292G CARs demonstrated a higher level of binding to bothhuman and murine ICAM-1 compared to non-transduced cells. However,despite increased binding of recombinant ICAM-1 to F292A CAR-expressingHEK 293T cells compared to their WT I domain-expressing counterparts(FIG. 1F), F292A CAR-Jurkat cells lacked any additional binding toplate-bound ICAM-1 compared to NT or WT I domain-expressing cells (FIG.1G). In the case of F265S I domain, which demonstrated solubleICAM-1-binding comparable to F292G (145 vs. 119 nM, Table 1), F265S CART cells failed to demonstrate any additional binding to plate-boundhuman ICAM-1 while elevated binding was more apparent to murine ICAM-1.As anticipated, T cells transduced to express R6.5 CAR, which isspecific to human ICAM-1 only, exhibited elevated binding to human butnot to murine ICAM-1 (FIG. 1G).

Example 14. Influence of CAR Affinity and Target Antigen Density on CART Cell Activation In Vitro

Jurkat T cells expressing I domain CARs were used to examine the extentto which CAR T cell activation was influenced by CAR affinity and ICAM-1antigen density in target cells. Jurkat T cells were incubated withvarious target cell lines with different ICAM-1 expression levels.ICAM-1 surface densities of target cell lines were estimated by firstassaying the levels of anti-ICAM-1 antibody binding to them andcomparing these signals to those obtained using 8 μm latex beads coupledwith known amounts of R6.5 antibody conjugated with cy5.5 (10³-10⁷antibodies per bead). The level of shift after incubation with R6.5(black) from non-labeled (grey) was used to estimate ICAM-1 density ineach indicated target cell line.

The panel of target cells include: HMEC-1 and bEnd.3, representing,respectively, healthy human and mouse cells with physiological levels ofICAM-1 (˜10⁴ molecules per cell); anaplastic thyroid carcinoma (8505C)expressing an intermediate level (˜10⁵ per cell); and cervical cancer(HeLa) cell lines expressing a high level of ICAM-1 (˜10⁶ per cell). Foradditional comparisons, we included 8505C with CRISPR/Cas9-mediatedICAM-1 gene inactivation (8505C/-ICAM-1) and 8505C treated with LPS toupregulate ICAM-1 expression (8505C/LPS). Table 3 summarizes ICAM-1 sitedensity in target cells used herein

TABLE 3 Target cells ICAM-1 density (molecules/cell) bEND.3 <10⁴ HMEC-1<10⁴ 8505C  10⁵ 8505C/LPS 10⁵-10⁶ 8505C/-ICAM-1 Non-detectable HeLa  10⁶

Activation of CAR T cells upon interaction with target cells wasexamined by measuring CD25 (IL-2 receptor α) and CD69 expression. CD25expression in Jurkat CAR T cells (WT, F292A, F292G, and TM) wereexamined after co-incubation with different target cell lines for 24 h(n=3-4). CD69 was induced after incubation with latex beads coated with10⁶ recombinant human ICAM-1-Fc molecules. Elevated levels of CD25 wereobserved in WT I domain CAR T cells following incubation withLPS-stimulated 8505C but not with other cell lines expressing lowerlevels of ICAM-1. In contrast, increased CD25 expression was seen whenhigh affinity TM CAR T cells were incubated with high ICAM-1 expressingcells as well as with HMEC-1 and bEnd.3 cells expressing basal levels ofICAM-1. A low-level of CD25 expression was detected on TM CAR T cellsfollowing incubation with target cells lacking ICAM-1 expression(8505C/-ICAM-1), likely due to homotypic cellular contacts mediated bymolecular interactions between TM CAR and basal expression of ICAM-1 onJurkat cells (˜10⁴ molecules/cell). T cells expressing F292G behavedsimilar to TM, except that CD25 expression was close to backgroundlevels following co-incubation with 8505C/-ICAM-1. The micromolaraffinity F292A T cells demonstrated selective activation displayingelevated CD25 expression only upon incubation with 8505C and 8505C/LPScells. This indicates that a threshold target antigen density of >10⁵ICAM-1 molecules per cell was required for F292A CAR T cell activation.In contrast to the ICAM-1 density-dependent activation of CD25,increased CD69 expression was observed even in the absence of targetcells, with expression levels aligning closely with CAR affinity toICAM-1, which was not further enhanced by incubation with ICAM-1 coatedlatex beads. Compared to CD25, CD69 induction appeared to require alower level threshold of antigen density for activation, which wasprovided by homotypic interaction between CAR T cells.

Example 15. Influence of CAR Affinity and Target Antigen Density on CART Cell Cytotoxicity In Vitro

After validating affinity and antigen-dependent activation ofCAR-modified Jurkat T cells, we sought to examine the influence of CARaffinity and antigen density on primary T cell activation andcytotoxicity in vitro. Primary T cells were transduced with TM, F292A,F292G, and WT I domain CARs, and added to various target cells todetermine their cytotoxic efficacy in vitro. Overall, there was apositive correlation between the rate of target cell lysis and ICAM-1expression (HeLa>8505C/LPS>8505C>HMEC-1>bEND.3) across all I domainvariant CAR T cells (FIG. 2A). The rate of killing was also faster whenT cells expressed CARs possessing higher affinity for ICAM-1(TM>F292G>F292A>WT).

To quantitatively compare the efficacy of killing by affinity variantCAR T cells, a variable slope sigmoidal curve (% live=100/[1+10^((t-τ)^(_) ^(50%)*Slope)]) was used to find the best fit values describing thetime required to achieve 50% killing (τ_50%) and the Hill slope (FIG.2B). The time to 50% target killing was longer with either loweraffinity CAR T cells or lower antigen density for the same CAR T cells.The Hill slope, corresponding to the rate of target killing by CAR Tcells, was higher with increases in affinity (lower Kd) for the sametarget cells. The Hill slope was also greater with increases in antigendensity for the same CAR T cells. CAR T cell killing of target cells wasspecific as evidenced by the lack of observed killing of ICAM-1 negative8505C cells by all of I domain variant CARs except TM. Low yet gradualkilling of 8505C/-ICAM-1 by TM T cells was likely due to cytotoxicactivation caused by homotypic cellular contacts mediated by TMinteraction with ICAM-1 in T cells. Table 4 summarizes time (hours) to50% killing determined by fitting data to a variable slope sigmoidalcurve.

TABLE 4 8505C/— 8505C/ CAR T HMEC bEND3 ICAM-1 8505C LPS HeLa WT n.d.n.d. n.d. n.d. n.d. 30.23 F292A n.d. n.d. n.d. 41.55 30.81 18.66 F292G21.05 16.23 n.d. 27.32 23.98 14.93 TM 13.45 13.03 32.63 17.12 15.0510.84 Only the best fit values with r-square values higher than 0.85 areshown; otherwise indicated as not-determined, n.d.

Table 5 shows Hill slope values determined by fitting data to a variableslope sigmoidal curve.

TABLE 5 8505C/— 8505C/ CAR T HMEC bEND3 ICAM-1 8505C LPS HeLa WT n.d.n.d. n.d. n.d. n.d. 0.09894 F292A n.d. n.d. n.d. 0.04424 0.04976 0.1096F292G 0.07538 0.05292 n.d. 0.06098 0.05872 0.1059 TM 0.08384 0.057930.05493 0.08686 0.08695 0.1099 Only the best fit values with r-squarevalues higher than 0.85 are shown; otherwise indicated asnot-determined, n.d.

ICAM-1 expression in primary T cells can be induced after T cellactivation such as by incubation with CD3/CD28 beads (˜10⁵molecules/cell). In comparison, WT CAR T cells possessing millimolaraffinity (Kd=1.5 mM) could specifically lyse HeLa cells only, indicatinga threshold antigen density of approximately 10⁶ molecules per cell for˜1 mM Kd CART cells. Importantly, F292A and WT I domain CAR T cells(Kd>10 μM) were unreactive to human and murine healthy control cells,HMEC-1 and b.END3 (˜10⁴ per cell; FIG. 2A).

IFN-γ release by CAR T cells aligned closely with the rate of targetcell death, where increasing levels were found in co-cultures containinghigher affinity CAR T cells and/or higher levels of target antigenexpression (FIG. 2D). One exception to target antigen density-dependentIFN-γ release was TM and F292G, which showed significant amounts ofIFN-γ release (>1 ng/ml) in the absence of target molecules(8505C/-ICAM-1). This is again likely due to the homotypic interactionsbetween T cells, which is also supported by the observation of thedifficulty with expanding TM CAR T cells, particularly when the level ofCAR expression was high. Release of IFN-γ by micromolar affinity CAR Tcells (F292A) was in proportion to the ICAM-1 density in target cells,demonstrated by a lack of release upon incubation with 8505C/-ICAM-1,and progressively increasing with incubation with HMEC-1, 8505C,8505C/LPS, and HeLa in this order (FIG. 2D). Consistent with WT Idomain's cytotoxicity toward HeLa cells, IFN-γ release upon incubationwith HeLa was comparable to the levels secreted by other higher affinityCAR T cells.

Example 16. In Vivo Efficacy of Affinity-Tuned I Domain CAR T Cells

We examined how affinity-dependent CAR T cell cytotoxicity patterns invitro would translate to tumor xenograft models in vivo. In solidtumors, CAR T cell efficacy is influenced by their ability to traffic totumor sites, penetrate, serially lyse tumor cells, and undergo expansionand contraction in accordance with tumor burden. Here, mice werexenografted by systemic i.v. injections of 0.75×10⁶ 8505C-FLuc⁺GFP⁺cells followed by treatment with ˜1-3×10⁶ I domain CAR T cells (WT,F292A, F265S, F292G, and TM), SSTR2-R6.5 CAR²⁹, NT (non-transduced) Tcells, and no T cells at 8-10 days post-xenograft (5-20% CARexpression). Tumor burden was evaluated by whole-body luminescenceimaging of firefly luciferase activity. Primary tumors localized to thelungs and liver with distant metastatic foci evident throughout the body(FIG. 3A). Cohorts receiving either no T cells or NT T cells succumbedto tumor burden within 3-4 weeks of tumor inoculation. Mice treated withTM CAR T cells displayed rapid initial reductions in tumor burden;however, at approximately 7 days post T cell injection, mice began toshow symptoms of systemic toxicity indicated by lethargy and weightloss, resulting in death by day 15 post treatment (FIGS. 3A-B). F292GCAR T cells were capable of tumor elimination with inconsistent toxicitydevelopment, which appeared to be partially dependent on tumor burden atthe time of CART cell treatment. For example, either delayed infusionsof F292G (119 nM affinity) CAR T cells (day 10) or higher tumor burdenat the time of treatment led to more frequent deaths. T cells expressingF265S (145 nM Kd) CARs, eliminated tumors without observable toxicity.This suggests that an I domain CAR affinity of ˜100 nM Kd defines anapproximate threshold affinity, above which (Kd less than 100 nM such1-10 nM) treatment leads to reduced discrimination between high and lowantigen densities and an increased likelihood of on-target off-tumortoxicity. Consistent with limited or lack of killing of 8505C by WT CART cells in vitro, tumor progression in vivo was unimpeded by thetreatment of WT CAR T cells, similar to NT T cells (FIG. 3B). Incontrast, F292A CAR T cells, which exhibited a much slower in vitro rateof 8505C killing compared to its higher affinity counterparts, achievedrapid reductions in tumor burden with no apparent toxicity irrespectiveof treatment timing (FIG. 3A-3B). Moreover, F292A CAR T in vivo efficacywas superior to the scFv-based R6.5 CAR despite >1,000-fold loweraffinity to ICAM-1 (10 nM vs. 20 as evidenced by a faster rate of tumorclearance and durable suppression of tumor relapse (FIG. 3A).

Overall, the anti-tumor efficacy of I domain CAR T cells led tostatistically significant increases in cohort survival compared with noT or NT T cell treated mice (FIG. 3C). However, CAR T cell-treated miceeven with no to little tumor burden began to show signs of toxicity(e.g., weight loss, loss of fur) that eventually led to frequent death˜10 weeks after T cell injections. This was suspected to be related tograft-versus-host disease³⁴ and not on-target, off-tumor toxicity assimilar toxicities were observed in mice treated with R6.5 CAR T cellsthat exclusively target human ICAM-1.

Example 17. Real-Time Imaging of CAR T Cell Kinetics, Efficacy, andToxicity

To spatiotemporally monitor T cell distribution in real-time by PET/CT,we introduced an imaging reporter gene, SSTR2 into the I domain CARvector using a ribosome skipping P2A sequence to ensure equal expressionof CAR and the reporter on the surface of T cells (FIG. 4A). Expressionof SSTR2 enabled binding and intracellular accumulation of an infused,positron-emitting, SSTR2-specific radiotracer, ¹⁸F-NOTA-Octreotide³⁰.Emitted signals were then detected with high resolution with no tissuepenetration issues by a micro PET scanner. Flow cytometry measurementsof SSTR2 reporter gene and Myc-tag expression representing CAR on thesurface of primary human T cells. Expression of SSTR2 and Myc tagged Idomain was confirmed by antibody staining by flow cytometry measurementsof SSTR2 reporter gene and Myc-tag expression representing CAR on thesurface of primary human T cells.

Mice were xenografted with 8505C tumors as before, and were treated withNT or F292A CAR T cells. Whole-body luminescence imaging was performedto estimate tumor burden while PET/CT imaging was performed on the sameday to track CAR T cell distribution (FIG. 4B). At each time point,blood was collected to measure human cytokines for correlation with Tcell dynamics. PET/CT images in mice displayed expected backgroundlevels in gall bladder, kidneys and bladder caused by radiotracerexcretion (FIG. 4B; far-right). In the NT treated control cohort, asmall but gradual increase in non-specific tracer uptake was observed,which was due to increasing tumor burden and the associated increase inblood pooling (FIG. 4B). In contrast, specific tracer uptake wasobserved in mice treated with SSTR2-F292A CAR T cells, demonstrating theexpansion and contraction phases in the lungs, with peak CAR T cellsignal occurring approximately at 22 days post xenograft, which is 4days following peak tumor burden (18 days post xenograft), and graduallydecreasing to background levels (FIGS. 4B-4C). This shows biphasic Tcell expansion and contraction phenomenon.

Cytokine analysis of serum obtained from treated mice demonstrated asurge in IFN-γ, IL-6, and CXCL10 concentrations prior to peak T cellexpansion, which also returned to background levels post tumorelimination and following contraction of T cell density in the lungs tobackground levels (FIG. 4D).

REFERENCES

-   1 Maher J, Brentjens R J, Gunset G, Riviere I, Sadelain M. Human    T-lymphocyte cytotoxicity and proliferation directed by a single    chimeric TCRzeta/CD28 receptor. Nat Biotechnol 20, 70-75 (2002).-   2. Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cell    receptor chimeric molecules as functional receptors with    antibody-type specificity. Proc Natl Acad Sci USA 86, 10024-10028    (1989).-   3. Hudecek M, et al. Receptor affinity and extracellular domain    modifications affect tumor recognition by ROR1-specific chimeric    antigen receptor T cells. Clin Cancer Res 19, 3153-3164 (2013).-   4. Watanabe K, et al. Target antigen density governs the efficacy of    anti-CD20-CD28-CD3 zeta chimeric antigen receptor-modified effector    CD8+ T cells. J Immunol 194, 911-920 (2015).-   5. Kochenderfer J N, et al. Eradication of B-lineage cells and    regression of lymphoma in a patient treated with autologous T cells    genetically engineered to recognize CD19. Blood 116, 4099-4102    (2010).-   6. Porter D L, Levine B L, Kalos M, Bagg A, June C H. Chimeric    antigen receptor-modified T cells in chronic lymphoid leukemia. New    England Journal of Medicine 365, 725-733 (2011).-   Grupp S A, et al. Chimeric antigen receptor-modified T cells for    acute lymphoid leukemia. N Engl J Med 368, 1509-1518 (2013).-   8. Brentjens R J, et al. CD19-targeted T cells rapidly induce    molecular remissions in adults with chemotherapy-refractory acute    lymphoblastic leukemia. Sci Transl Med 5, 177ra138 (2013).-   9. Brudno J N, Kochenderfer J N. Toxicities of chimeric antigen    receptor T cells: recognition and management. Blood 127, 3321-3330    (2016).-   10. Cheever M A, et al. The prioritization of cancer antigens: a    national cancer institute pilot project for the acceleration of    translational research. Clin Cancer Res 15, 5323-5337 (2009).-   11. Kakarla S, Gottschalk S. CAR T cells for solid tumors: armed and    ready to go? Cancer J 20, 151-155 (2014).-   12. Lamers C H, et al. Treatment of metastatic renal cell carcinoma    with autologous T-lymphocytes genetically retargeted against    carbonic anhydrase IX: first clinical experience. J Clin Oncol 24,    e20-22 (2006).-   13. Parkhurst M R, et al. T cells targeting carcinoembryonic antigen    can mediate regression of metastatic colorectal cancer but induce    severe transient colitis. Mol Ther 19, 620-626 (2011).-   14. Morgan R A, Yang J C, Kitano M, Dudley M E, Laurencot C M,    Rosenberg S A. Case report of a serious adverse event following the    administration of T cells transduced with a chimeric antigen    receptor recognizing ERBB2. Mol Ther 18, 843-851 (2010).-   15. Tian S, Maile R, Collins E J, Frelinger J A. CD8+ T cell    activation is governed by TCR-peptide/MHC affinity, not dissociation    rate. J Immunol 179, 2952-2960 (2007).-   16. Hebeisen M, Allard M, Gannon P O, Schmidt J, Speiser D E,    Rufer N. Identifying Individual T Cell Receptors of Optimal Avidity    for Tumor Antigens. Front Immunol 6, 582 (2015).-   17. Zhong S, et al. T-cell receptor affinity and avidity defines    antitumor response and autoimmunity in T-cell immunotherapy. Proc    Natl Acad Sci USA 110, 6973-6978 (2013).-   18. Liu X, et al. Affinity-Tuned ErbB2 or EGFR Chimeric Antigen    Receptor T Cells Exhibit an Increased Therapeutic Index against    Tumors in Mice. Cancer Res 75, 3596-3607 (2015).-   19. Caruso H G, et al. Tuning Sensitivity of CAR to EGFR Density    Limits Recognition of Normal Tissue While Maintaining Potent    Antitumor Activity. Cancer Res 75, 3505-3518 (2015).-   20. Arcangeli S, et al. Balance of Anti-CD123 Chimeric Antigen    Receptor Binding Affinity and Density for the Targeting of Acute    Myeloid Leukemia. Mol Ther, (2017).-   21. Chmielewski M, Hombach A, Heuser C, Adams G P, Abken H. T cell    activation by antibody-like immunoreceptors: increase in affinity of    the single-chain fragment domain above threshold does not increase T    cell activation against antigen-positive target cells but decreases    selectivity. J Immunol 173, 7647-7653 (2004).-   22. Schmid D A, et al. Evidence for a TCR affinity threshold    delimiting maximal CD8 T cell function. J Immunol 184, 4936-4946    (2010).-   23. Corse E, Gottschalk R A, Krogsgaard M, Allison J P. Attenuated T    cell responses to a high-potency ligand in vivo. PLoS Biol 8,    (2010).-   24. Park S, et al. Tumor suppression via paclitaxel-loaded drug    carriers that target inflammation marker upregulated in tumor    vasculature and macrophages. Biomaterials 34, 598-605 (2013).-   25. Dustin M L, Rothlein R, Bhan A K, Dinarello C A, Springer T A.    Induction by IL 1 and interferon-gamma: tissue distribution,    biochemistry, and function of a natural adherence molecule (ICAM-1).    J Immunol 137, 245-254 (1986).-   26. Shimaoka M, et al. Reversibly locking a protein fold in an    active conformation with a disulfide bond: integrin alphaL I domains    with high affinity and antagonist activity in vivo. Proc Natl Acad    Sci USA 98, 6009-6014 (2001).-   27. Jin M, et al. Directed evolution to probe protein allostery and    integrin I domains of 200,000-fold higher affinity. Proc Natl Acad    Sci USA 103, 5758-5763 (2006).-   28. Wong R, Chen X, Wang Y, Hu X, Jin M M. Visualizing and    Quantifying Acute Inflammation Using ICAM-1 Specific Nanoparticles    and MRI Quantitative Susceptibility Mapping. Ann Biomed Eng 40,    1328-1338 (2011).-   29. Vedvyas Y, et al. Longitudinal PET imaging demonstrates biphasic    CAR T cell responses in survivors. JCI Insight 1, e90064 (2016).-   30. Laverman P, et al. A novel facile method of labeling octreotide    with (18)F-fluorine. J Nucl Med 51, 454-461 (2010).-   31. McBride W J, et al. A novel method of 18F radiolabeling for PET.    J Nucl Med 50, 991-998 (2009).-   32. Kinahan P E, Fletcher J W. Positron emission tomography-computed    tomography standardized uptake values in clinical practice and    assessing response to therapy. Semin Ultrasound CT MR 31, 496-505    (2010).-   33. Leelawattanachai J, Kwon K W, Michael P, Ting R, Kim J Y, Jin    M M. Side-by-Side-   Comparison of Commonly Used Biomolecules That Differ in Size and    Affinity on Tumor Uptake and Internalization. PLoS One 10, e0124440    (2015).-   34. Poirot L, et al. Multiplex Genome-Edited T-cell Manufacturing    Platform for “Off-the-Shelf” Adoptive T-cell Immunotherapies. Cancer    Res 75, 3853-3864 (2015).-   35. Kang S, et al. Virus-mimetic polyplex particles for systemic and    inflammation-specific targeted delivery of large genetic contents.    Gene Ther 20, 1042-1052 (2013).-   36. Hinrichs C S, Restifo N P. Reassessing target antigens for    adoptive T-cell therapy. Nat Biotechnol 31, 999-1008 (2013).-   37. Newick K, O'Brien S, Moon E, Albelda S M. CAR T Cell Therapy for    Solid Tumors. Annu Rev Med 68, 139-152 (2017).-   38. Ledebur H C, Parks T P. Transcriptional regulation of the    intercellular adhesion molecule-1 gene by inflammatory cytokines in    human endothelial cells. Essential roles of a variant NF-kappa B    site and p65 homodimers. J Biol Chem 270, 933-943 (1995).-   39. Usami Y, et al. Intercellular adhesion molecule-1 (ICAM-1)    expression correlates with oral cancer progression and induces    macrophage/cancer cell adhesion. Int J Cancer 133, 568-578 (2013).-   40. Roland C L, Harken A H, Sarr M G, Barnett C C, Jr. ICAM-1    expression determines malignant potential of cancer. Surgery 141,    705-707 (2007).-   41. Guo P, et al. ICAM-1 as a molecular target for triple negative    breast cancer. Proc Natl Acad Sci USA 111, 14710-14715 (2014).-   42. Carman C V, Springer T A. Integrin avidity regulation: are    changes in affinity and conformation underemphasized? Curr Opin Cell    Biol 15, 547-556 (2003).-   43. Boissonnas A, Fetler L, Zeelenberg I S, Hugues S, Amigorena S.    In vivo imaging of cytotoxic T cell infiltration and elimination of    a solid tumor. J Exp Med 204, 345-356 (2007).-   44. Porter B B, Harty J T. The onset of CD8+-T-cell contraction is    influenced by the peak of-   Listeria monocytogenes infection and antigen display. Infect Immun    74, 1528-1536 (2006).-   45. Keu K V, et al. Reporter gene imaging of targeted T cell    immunotherapy in recurrent glioma. Sci Transl Med 9, (2017).-   46. Yaghoubi S S, et al. Noninvasive detection of therapeutic    cytolytic T cells with 18F-FHBG PET in a patient with glioma. Nat    Clin Pract Oncol 6, 53-58 (2009).-   47. Drent E, et al. A Rational Strategy for Reducing On-Target    Off-Tumor Effects of CD38-Chimeric Antigen Receptors by Affinity    Optimization. Mol Ther, (2017).-   48. Kalergis A M, et al. Efficient T cell activation requires an    optimal dwell-time of interaction between the TCR and the pMHC    complex. Nat Immunol 2, 229-234 (2001).-   49. Valitutti S. The Serial Engagement Model 17 Years After: From    TCR Triggering to Immunotherapy. Front Immunol 3, 272 (2012).-   50. McMahan R H, McWilliams J A, Jordan K R, Dow S W, Wilson D B,    Slansky J E. Relating-   TCR-peptide-MHC affinity to immunogenicity for the design of tumor    vaccines. The Journal of clinical investigation 116, 2543-2551    (2006).-   51. Robbins P F, et al. Single and dual amino acid substitutions in    TCR CDRs can enhance antigen-specific T cell functions. J Immunol    180, 6116-6131 (2008).-   52. Co M S, Deschamps M, Whitley R J, Queen C. Humanized antibodies    for antiviral therapy. Proc Natl Acad Sci USA 88, 2869-2873 (1991).-   53. Leelawattanachai J, Kwon K-W, Michael P, Ting R, Kim J-Y, Jin    M M. Side-by-Side Comparison of Commonly Used Biomolecules That    Differ in Size and Affinity on Tumor Uptake and Internalization.    PLoS One 10, e0124440 (2015).-   54. Jin M, et al. Directed evolution to probe protein allostery and    integrin I domains of 200,000-fold higher affinity. Proc Natl Acad    Sci USA 103, 5758-5763 (2006).-   55. Wong R, Chen X, Wang Y, Hu X, Jin M M. Visualizing and    quantifying acute inflammation using ICAM-1 specific nanoparticles    and MRI quantitative susceptibility mapping. Ann Biomed Eng 40,    1328-1338 (2012).

What is claimed is:
 1. A Chimeric antigen receptor (CAR) comprising fromN-terminus to C-terminus: (i) an I domain of the α_(L) subunit of humanlymphocyte function-associated antigen-1, (ii) a transmembrane domain,(iii) at least one co-stimulatory domains, and (iv) an activatingdomain.
 2. The CAR of claim 1, wherein the I domain is a wild-type Idomain or a mutant thereof having 1 to 3 amino acid mutations.
 3. TheCAR of claim 1, wherein the I domain binds ICAM-1 at an affinity betweenabout 1 mM to about 1 nM.
 4. The CAR of claim 2, wherein the wild-type Idomain comprises the sequence of 130-310 amino acids of SEQ ID NO:
 1. 5.The CAR of claim 4, wherein the mutant has one or more mutations at theamino acid residue 265, 288, 289, 292, 295, or 309 of the wild-type Idomain, or a sequence having at least 95% identity thereof, wherein thenumbering of the amino acid residues corresponds to the amino acidresidues of SEQ ID NO:
 1. 6. The CAR of claim 5, wherein the mutant hasone or more mutations of I288N, I309T, L295A, F292A, F292S, L289G,F292G, and F265S,
 7. The CAR of claim 5, wherein the mutant has amutation of F265S/F292G, or F265S/F292G/G311C.
 8. The CAR according toclaim 1, wherein the co-stimulatory domain is selected from the groupconsisting of CD28, 4-1BB, ICOS-1, CD27, OX-40, GITR, and DAP10.
 9. TheCAR according to claim 1, wherein the activating domain is CD3 zeta. 10.An isolated nucleic acid sequence encoding the CAR of claim
 1. 11. Tcells or natural killer cells modified to express the CAR of claim 1.12. An adoptive cell therapy method for treating cancer, comprising thesteps of: administering the CAR-T cells of claim 11 to a subjectsuffering from cancer, wherein the cancer cells of the subjectoverexpress ICAM-1, and the CAR T cells bind to the cancer cells to killthe cancer cells.
 13. The method according to claim 12, wherein thecancer is thyroid cancer, gastric cancer, pancreatic cancer, or breastcancer.